Metabonomics is a rapidly growing area of scientific research. It is a systems approach for studying in vivo metabolic profiles and can provide information on a particular disease, toxicity, and gene function. In metabonomics, the effect of a pharmaceutical candidate on a whole animal or organism is investigated by studying the changes in metabolism over a time course following compound administration. The analytical data generated in these studies is analyzed by multi-variant mathematical techniques such as Principle Component Analysis (hereinafter “PCA”). This mathematical technique is employed to highlight both subtle and gross differences in the samples being examined.
To date, the vast majority of work in this field has utilized proton-nuclear magnetic resonance (NMR) as the analytical method of choice. While being very effective, NMR has several disadvantages, namely, poor sensitivity, time consuming analysis, and the non-detection of some chemical classes, e.g., sulfates. A further draw back to NMR is that as all of the signals are contained in one spectrum it is easy for one large compound to mask subtle but important changes in a low concentration analyte. Further, the necessary removal of xenobiotic-related compounds from the NMR spectrum also removes signals from endogenous compounds of interest thus reducing the data set used for subsequent PCA analysis.
Chromatography, both gaseous and liquid, combined with spectrometric analysis such as ultraviolet spectroscopy, infrared spectroscopy, nuclear magnetic resonance or mass spectrometry, has evolved into a powerful technique. Electrospray mass spectrometry coupled with liquid chromatography (LC/MS) has become the technique of choice for bioanalysis, both quantitative and qualitative. The technique is robust, sensitive, and selective with sensitivities up to the pg/mL range readily achieved. The use of short columns and rapid gradients has also allowed LC/MS to achieve analytical cycle times in the region of one sample per minute with good chromatographic resolution and sensitivity. Thus the application of LC/MS to metabonomics is a logical step. While the mass spectrometer will give both high sensitivity quantitation and structural information, the chromatography step will address the issue of overlapping signals by separating out the analyte giving a time resolution. The use of LC/MS is preferred over mass spectrometry infusion as the chromatography step reduces ion suppression by reducing the number of competing ions going into the mass spectrometer at any given time. Another advantage of LC/MS over NMR is observed in data analysis. When removing xenobiotic-related material only a small time slice at one or two specific masses will be removed from the data set. Therefore, the remaining LC/MS data is left unaltered and available for mathematical analysis, e.g., PCA.
Principle component analysis is a very effective mathematical device for analyzing the data obtained from MS. However, PCA is a two-dimensional technique whereas data obtained from LC/MS is three-dimensional. Currently, there exists a need to enable PCA to maintain the chromatographic separation information that is normally lost in conventional PCA analysis.
In general, there are many experimental conditions where the information about the results is contained in data that is characterized by multiple parameters. When the interpretive tools cannot handle these parameters, information is lost. A methodology that improves the resolution of analysis of LC/MS data that is analyzed by the PCA process is applicable to other data sets.